Formation of N-alkylated protoporphyrin IX in the livers of mice after diethylnitrosamine treatment.

The administration of di[1-14C]ethylnitrosamine to phenobarbital-pretreated mice resulted in the formation of a radiolabelled green pigment in their livers. Green-pigment concentrations were time- and dose-dependent, maximum levels being reached 1-2 h after dosing. There was only a slight decrease in cytochrome P-450 levels and accumulation of porphyrins in the liver at this time. Dimethyl- or dipropyl-nitrosamine also caused an accumulation of similar, though not identical, compounds in the liver. The formation of green pigment was induced by pretreatment of mice with phenobarbital or 3-methylcholanthrene and was inhibited by the acute administration of pyrazole or ethanol. From the absorption spectra, the green pigment methyl esters appeared to be N-alkylporphyrins. Analysis of the diethylnitrosamine-induced green pigment by high-pressure liquid chromatography showed it to be more polar than the expected N-ethylprotoporphyrin IX, having a retention time similar to that of N-hydroxyethylprotoporphyrin IX. Desorption chemical-ionization mass spectrometry gave a protonated molecular ion, m/z 635, compatible with N-hydroxyethylprotoporphyrin IX. The presence of a free hydroxy group was demonstrated by acetylation with [1-14C]acetic anhydride. No conversion of N-ethylprotoporphyrin IX into N-hydroxyethylprotoporphyrin IX could be demonstrated in vivo or in vitro. Little or no N-ethylprotoporphyrin IX accumulated in the livers of mice given diethylnitrosamine. It was concluded that N-hydroxyethylprotoporphyrin IX is the primary reaction product between an active metabolite of diethylnitrosamine and hepatic haem.     

Impaired glucose transport in inguinal adipocytes after short-term high-sucrose feeding in mice

Diets enriched in sucrose severely impair metabolic regulation and are associated with obesity, insulin resistance and glucose intolerance. In the current study, we investigated the effect of 4 weeks high-sucrose diet (HSD) feeding in C57BL6/J mice, with specific focus on adipocyte function. Mice fed HSD had slightly increased adipose tissue mass but displayed similar hepatic triglycerides, glucose and insulin levels, and glucose clearance capacity as chow-fed mice. Interestingly, we found adipose depot-specific differences, where both the non- and insulin-stimulated glucose transports were markedly impaired in primary adipocytes isolated from the inguinal fat depot from HSD-fed mice. This was accompanied by decreased protein levels of both GLUT4 and AS160. A similar but much less pronounced trend was observed in the retroperitoneal depot. In contrast, both GLUT4 expression and insulin-stimulated glucose uptake were preserved in adipocytes isolated from epididymal adipose tissue with HSD. Further, we found a slight shift in cell size distribution towards larger cells with HSD and a significant decrease of ACC and PGC-1α expression in the inguinal adipose tissue depot. Moreover, fructose alone was sufficient to decrease GLUT4 expression in cultured, mature adipocytes.Altogether, we demonstrate that short-term HSD feeding has deleterious impact on insulin response and glucose transport in the inguinal adipose tissue depot, specifically. These changes occur before the onset of systemic glucose dysmetabolism and therefore could provide a mechanistic link to overall impaired energy metabolism reported after prolonged HSD feeding, alone or in combination with HFD.     

Palmitoylation of LAT contributes to its subcellular localization and stability

Palmitoylation is a protein modification for trafficking to lipid raft. Without palmitoylation, linker for activation of T cells (LAT), an adaptor molecule mediating T cell receptor signaling, is unable to localize in lipid rafts and to mediate T cell activation. We here show a novel role for palmitoylation in LAT trafficking to the plasma membrane and in the stability of the LAT protein. The human LAT mutant lacking palmitoylation was unable to traffic to the plasma membrane despite the presence of transmembrane portion. The mouse LAT mutant lacking palmitoylation was unstable and susceptible to degradation via the proteasome pathway. The human LAT mutant became unstable when the extracellular portion was swapped for that from mouse, indicating that both palmitoylation and the extracellular portion regulate the stability of LAT. These results suggest that palmitoylation has an important role in trafficking to the plasma membrane and the stability of LAT.     

Cellular and subcellular localization of catalase in the heart of transgenic mice.

Previous studies have described a cardiac-specific, catalase-overexpressing transgenic mouse model that was used to study myocardial oxidative injury. This study was undertaken to demonstrate cellular and subcellular localization of catalase in the hearts of transgenic mice. By the light microscopic immunoperoxidase method, we found that the overexpressed catalase was exclusively localized in cardiomyocytes. The ratios of immunoreactive cardiomyocytes in the heart were quite different among three transgenic lines examined but agreed with the elevated levels of catalase activity. In the cardiac blood vessels, positive cells were found in the walls of pulmonary veins and the vena cava, which consist of cardiomyocytes, but not in the pulmonary arteries, aorta, or cardiac valves. The electron microscopic immunogold method revealed that the elevated catalase was in sarcoplasm, nucleus, and peroxisomes, but not in mitochondria. In contrast to these distributions, catalase in the non-transgenic cardiomyocytes was in peroxisomes only. In addition, the number and size of peroxisomes in the transgenic cardiomyocytes were markedly increased, but no other ultrastructural changes were observed in comparison with those of non-transgenic mice. These results demonstrated that the elevated catalase in transgenic mouse heart is localized in cardiomyocytes and is distributed to peroxisomal and extraperoxisomal, but not mitochondrial, compartments.     

Comparison of acute responses of mice livers to short-term exposure to nano-sized or micro-sized silver particles

Mice were fed either 13 nm silver nanoparticles or 2–3.5 μm silver microparticles. The livers were then obtained after 3 days and subjected to a histopathological analysis. The nanoparticle-fed and microparticle-fed livers both exhibited lymphocyte infiltration in the histopathological analysis, suggesting the induction of inflammation. In vitro, a human hepatoma cell line (Huh-7) was treated with the same silver nanoparticles and microparticles. The mitochondrial activity and glutathione production were hardly affected. However, the DNA contents decreased 15% in the nanoparticle-treated cells and 10% in the microparticle-treated cell, suggesting a more potent induction of apoptosis by the nanoparticles. From a microarray analysis of the RNA from the livers of the nano- and micro-particle-fed mice, the expression of genes related to apoptosis and inflammation was found to be altered. These gene expression changes in the nanoparticle-treated livers lead to phenotypical changes, reflecting increased apoptosis and inflammation. The changes in the gene expression were confirmed by using a semi-quantitative RT-PCR.     

Protoporphyrin-induced Photohemolysis : Differences related to the subcellular distribution of protoporphyrin in erythropoietic protoporphyria and when added to normal red cells

• 1.1. When protoporphyrin is added to normal red cells it distributes to about 30% in the stroma and 70% in the cytosol. By comparison, in erythropoietic protoporphyria red cell protoporphyrin is found to more than 95% in the cytosol.
• 2.2. At equimolar concentrations of protoporphyrin the photohemolysis is much more severe in normal red cells with exogenous protoporphyrin than in red cells from patients with erythropoietic protoporphyria.
• 3.3. The photohemolysis is markedly enhanced when D₂O is used as solvent instead of H₂O.
• 4.4. The results suggest that the photodamage is determined by the ability of susceptible structures to accumulate porphyrins, the partition of porphyrins between lipophilic and hydrophilic structures and the longevity of singlet oxygen in lipophilic environments.

     

CK2 phosphorylates TNFAIP1 to affect its subcellular localization and interaction with PCNA

TNFAIP1 is a protein which can be induced by tumor necrosis factorα (TNFα) and interleukin-6 (IL-6), it may play roles in DNA synthesis, DNA repair, cell apoptosis and human diseases. However, very little has been known about how TNFAIP1 acts in these physiological processes. In this paper, CK2β was identified as a partner of TNFAIP1 by screening the HeLa cDNA library in yeast two-hybrid system with TNFAIP1 as a bait. Furthermore, it was demonstrated that CK2 could phosphorylate TNFAIP1 in vitro and in vivo, which facilitated the distribution of TNFAIP1 in nucleus and enhanced its interaction with PCNA. It is suggested that the phosphorylation of TNFAIP1 may be required for its functions.     

Expression and subcellular localization of a membrane protein related to Hsp30p in Saccharomyces cerevisiae

The Saccharomyces cerevisiae YDR033w gene product is homologous to Hsp30p and Yro2p, both of which are induced during heat shock. To investigate the subcellular localization of the YDR033w gene product, hemagglutinin (HA) epitope-tagged protein was expressed, detected on immunoblots, and localized by immunofluorescence to cell membranes, primarily the plasma membrane. A punctuate immunofluorescence pattern was observed within cell buds. The nuclear envelope, but not the vacuole or mitochondrial membranes, were also immunostained. We refer to YDR033w as MRH1 to denote that it encodes a membrane protein related to Hsp30p.     

The subcellular localization of ubiquinone in human neutrophils.

Copper and zinc K-edge-extended X-ray-absorption fine structures were measured for the metal sites of freeze-dried bovine superoxide dismutase and the model compounds tetrakis(imidazole)cupric nitrate and tetrakis(imidazole)zinc perchlorate. Detailed simulation of the spectra indicates that the copper site of the enzyme is best fit by co-ordination of four imidazole groups with Cu-N(alpha) distances of 0.198 nm (1.98 A). The zinc site is best fit by three imidazole groups at 0.201 nm (2.01 A) and an oxygen (from aspartate) at 0.203 nm (2.03 A).     

Ribosome function in livers of porphyric mice.

1. A porphyrinogenic drug, 3,5-diethoxycarbonyl-1,4-dihydrocollidine, caused a decrease in the proportion of single ("run off") ribosomes, and an increase in the number of polyribosomes, in the livers of treated animals. 2. No change could be detected in the distribution of amino acid incorporation among hepatic polyribosomes.     

Fetal lung growth after short-term tracheal occlusion is linearly related to intratracheal pressure.

Prenatal tracheal occlusion (TO) has been shown to accelerate fetal lung growth, yet the mechanism is poorly understood. The goal of this study was to determine the relationship between fetal intratracheal pressure (Pitr) and fetal lung growth after TO. Fetal lambs underwent placement of an intratracheal catheter and a reference catheter at 115--120 days gestation (term, 145 days). Fetal Pitr was continuously controlled at three levels (high, 8 mmHg; moderate, 4 mmHg; low, 1 mmHg) by a servo-regulated pump. The animals were killed after 4 days, and the parameters of lung growth were compared. Lung volume (136.0 +/- 16.7, 94.9 +/- 9.7, 55.5 +/- 12.4 ml/kg), lung-to-body weight ratio (6.31 +/- 0.70, 4.89 +/- 0.38, 3.39 +/- 0.22%), whole right lung dry weight (3.01 +/- 0.29, 2.53 +/- 0.15, 2.07 +/- 0.24 g/kg), right lung DNA (130.0 +/- 11.3, 116.7 +/- 8.6, 97.5 +/- 10.9 mg/kg), and protein contents (1,865.5 +/- 92.5, 1,657.6 +/- 106.8, 1,312.0 +/- 142.5 mg/kg) in high, moderate, and low groups, respectively, all increased in the moderate compared with the low group and increased further in the high compared with the moderate group. Morphometry confirmed a stepwise increase in the volume of respiratory region and alveolar surface area. We conclude that lung growth in the first 4 days after TO is closely correlated with fetal Pitr, offering additional evidence that an increase in lung expansion is one of the major factors responsible for TO-induced lung growth.     

Differential subcellular localization of zinc in the rat retina.

In the retina, zinc is believed to be a modulator of synaptic transmission and a constituent of metalloenzymes. To determine whether the intracellular localization of zinc correlates with function, we examined the localization of endogenous zinc in the rat retina using the silver amplification method. By light microscopy, reaction products were detected in the pigment epithelial cells (PE), the inner segment of photoreceptors (IS), the outer nuclear layer (ONL) and the inner nuclear layer (INL), the outer plexiform layer (OPL) and the inner plexiform layer (IPL), and the ganglion cell layer (GC). The heaviest accumulation of precipitate was observed in PE and IS, whereas only a little precipitate was found in GC. When the intracellular zinc was chelated with diethyldithiocarbamate, a small amount of precipitate was observed only in ONL. By electron microscopy, zinc was associated with three compartments. In OPL and IPL, zinc was associated with neural processes, while in PE, IS, INL, and GC it was associated with the Golgi apparatus. In ONL, zinc was associated with the nucleus. Zinc in the neural processes is believed to act as a modulator of synaptic transmission, and zinc associated with the Golgi apparatus is assumed to catalyze metalloenzyme reactions.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Iodination by stimulated human neutrophils. Studies on its stoichiometry, subcellular localization and relevance to microbial killing

Myeloperoxidase of phagocytic leucocytes is thought to utilize H2O2 to oxidize halides, which then react with and kill ingested microbes. This hypothesis was based largely on the incorporation of radiolabelled iodide into cells that had phagocytosed bacteria. The present studies investigated the stoichiometry of these reactions and the subcellular localization and electrophoretic pattern of the cellular components that became iodinated. 1. The stoichiometry of the reactions are such that only a small proportion (less than 0.3%) of the total oxygen consumed is utilized for iodination. Iodination after stimulation with the soluble stimulus phorbol myristate acetate (PMA), which is not known to involve the azurophil granules and their contained myeloperoxidase, was comparable with that occurring after bacterial ingestion. 2. Analytical subcellular fractionation of cells that had phagocytosed bacteria localized about 25% of the radioactivity to the membranes, and most of the residual radioactivity distributed with the bacteria and dense granules. In cells stimulated with PMA, more of the radioactivity was associated with the membranes, but about half was still associated with the dense granules. 3. Autoradiographs after dodecyl sulphate/polyacrylamide-gel electrophoresis of cells stimulated with opsonized bacteria gave a similar distribution of iodinated components to that obtained with cells that had been stimulated with PMA or iodinated with Iodogen. These patterns of iodination were very different from those obtained when bacteria alone were iodinated with Iodogen or myeloperoxidase and H2O2. Preparations in which bacteria had been phagocytosed did not show evidence of iodination of bacterial proteins or coating opsonins. Thus positive evidence for the iodination of bacteria has not been produced, and the role of iodination in the microbicidal process of neutrophils remains to be established.     

Mutational analysis of MukE reveals its role in focal subcellular localization of MukBEF

Bacterial condensin MukBEF is essential for global folding of the Escherichia coli chromosome. MukB, a SMC (structural maintenance of chromosome) protein, comprises the core of this complex and is responsible for its ATP‐modulated DNA binding and reshaping activities. MukF serves as a kleisin that modulates MukB–DNA interactions and links MukBs into macromolecular assemblies. Little is known about the function of MukE. Using random mutagenesis, we generated six loss‐of‐function point mutations in MukE. The surface mutations clustered in two places. One of them was at or close to the interface with MukF while the other was away from the known interactions of the protein. All loss‐of‐function mutations affected focal localization of MukBEF in live cells. In vitro, however, only some of them interfered with the assembly of MukBEF into a complex or the ability of MukEF to disrupt MukB–DNA interactions. Moreover, some MukE mutants were able to join intracellular foci formed by endogenous MukBEF and most of the mutants were efficiently incorporated into MukBEF even in the presence of endogenous MukE. These data reveal that focal localization of MukBEF involves other activities besides DNA binding and that MukE plays a central role in them.